In the analysis of complex substance mixtures of biological and/or chemical origin, the analyst not only has the task of identifying the structure of individual substances present in the mixture, but also has the problem every time of capturing all substances present in the mixture and quantifying them if at all possible. This should proceed very rapidly and with high precision, i.e. with a small error deviation. This becomes all the more important when information is to be obtained on a biological system, for example on a microorganism grown under certain fermentation conditions or on a plant grown under different environmental conditions or on a wild type organism such as a microorganism or a plant in comparison to its genetically modified mutant. Such comparisons are necessary in order to enable assignment of mutations of unknown genes in the genome of these organisms to a certain metabolic phenotype.
The success in the analysis of these substance mixtures, for example chemical synthesis mixtures, from combinatorial chemistry or from extracts from microorganisms, plants or plant parts depends to a great extent upon the rapidity and reproducibility of the analysis used. In such a screening, a multitude of samples have to be scanned through; rapid, simple, highly sensitive and highly specific analytical processes are therefore required.
A main problem of this analysis is the rapid, simple, reproducible and quantifiable identification of the substances present in the mixtures. In general, the products are analyzed using separation processes such as thin-layer chromatography (=TLC), high-pressure liquid chromatography (=HPLC) or gas chromatography (=GC). However, it is not possible with the aid of these chromatographic processes to rapidly and simply identify and quantify a wide range of substances. Processes such as NMR or mass spectrometry have also been described for this task. However, a certain degree of preparation of the samples is generally required for these analytical processes, such as workup via, for example, salt precipitation and/or subsequent chromatography, concentration, desalting of the samples, buffer exchange or removal of any detergents present in the sample.
After this pretreatment, the samples can be used for the aforementioned analyses and it is possible to identify and quantify individual substances in selected samples. However, these processes are time-consuming and only permit a limited sample throughput, so that such analytical processes do not find use in high-throughput screening (=HTS) or the broad screening of substance mixtures in biological or chemical samples. An advantage in very precise methods such as NMR or IR spectroscopy is that they provide information both on the structure and, in some cases, on the quantity of a substance.
In order to enable higher sample throughput in HTS, indirect, readily measurable processes such as color reactions in the visible region, cloudiness measurements, fluorescence, conductivity measurements, etc. are used in many cases. Although they are in principle very sensitive, they are also prone to faults. Disadvantages in this case are in particular that many falsely positive samples are analyzed in this procedure, and that, since they are indirect detection processes, there is no information about the structure and/or the quantity of a compound. In order to be able to exclude these false positives in the further procedure, further analytical processes, for example NMR, IR, HPLC-MS or GC-MS, are generally used after a first rapid analysis. This is again very time-consuming.
Generally, it can be stated that the improvement in the sensitivity and the conclusiveness of the detection processes leads to a decrease in the speed of an analysis.
When working with complex biological mixtures, for example extracts from microorganisms, plants and/or animals, it also has to be taken into account that individual compounds are present in the mixtures only in very small amounts or only small amounts of the individual sample itself are available for the analysis, so that the method used has to have a high sensitivity. Moreover, the involatile buffers and/or salts frequently present in biological samples constitute a problem for some analysis methods, since they adversely affect the sensitivity of the methods or indeed their use. The same applies to the presence of detergents in these samples.
For the analysis of complex sample mixtures, the prior art discloses mass spectrometry processes which range, for example, from the analysis of samples from synthetic chemistry, petrochemistry, environmental samples and biological material. However, these methods are used only for the analysis of individual known compounds in these samples. Wide measurement ranges, for example in the context of an HTS or in the identification and quantification of a multitude of compounds in these samples, are not described.
One method that finds use for substances which are extractable from the substance mixtures and are volatile is the coupling of gas chromatography and mass spectrometry (=GC-MS). For the analysis of substances or analytes which cannot easily be transferred to the gas phase or only with difficulty and for which a large excess of solvent present has to be removed, liquid chromatography- or high-pressure liquid chromatography-mass spectrometry (=HPLC-MS) is used. A review of the different LC-MS methods and their equipment can be taken from the publication of Niessen et al. (Journal of Chromatography A, 703, 1995: 37–57). The US documents U.S. Pat. No. 4,540,884 and U.S. Pat. No. 5,397,894 describe and claim mass spectrometers and their construction.
With the aid of the aforementioned methods, it is possible to determine substances in a molecular weight range of up to 100 kD (=kilodaltons), i.e. it is possible to determine a wide range of substances, for example in a lower mass range of up to about 5000 D (=daltons) such as fatty acids, amino acids, carboxylic acids, oligo- or polysaccharides, steroids, etc., and/or in a higher mass range above 5000 D such as peptides, proteins, oligonucleotides and oligosaccharides or other polymers. It is also possible to analyze high molecular weight materials such as coal tar, humic acid, fulvic acid or kerogens (Zenobie and Kno-chenmuss, Mass Spec. Rev., 1998, 17, 337–366). It is possible to determine both the identity and the structure of substances, although the structural analysis is not always unambiguous, so that it has to be confirmed using other methods, for example NMR.
G. Hopfgartner and F. Vilbois (Analysis, 2001, 28, No. 10, 906–914) describe a process for screening with the aid of LC-MS of metabolites, formed in vitro or in vivo, of compounds of known structure which are as active ingredients in different phases of the active ingredient development. This process proceeds in two steps. In the first search step, ions of interest are captured in a rapid“full scan mode”, said ions being possible candidates for the further investigations. They may be ions which correspond to ions of particularly high intensity or be candidates of possible decomposition products or metabolites of the active ingredients. These ions are used in a second scan for identifying the chemical structure of these ions or compounds after a fragmentation in a collision chamber of the mass spectrometer. In order to enable rapid elucidation of the ion or metabolite structure, the collision chamber always contains collision gas. A disadvantage in the structural determination is that a known mass of a precursor ion, of a fragment or of an ion adduct is required. Advantageously, the starting structure of the substance to be investigated should be known for the HPLC-MS in these experiments. Since HPLC-MS alone is unsuitable for absolute structural determination, but the structure of the starting compound is known, it is possible to make statements about the structure of any metabolites. Since the structure of the substance which is to be developed as an active ingredient is known, statements can be made about the structure of the unknown metabolites of the active ingredient with some certainty. However, the statement is complicated or prevented by possible overlappings of other compounds of the same mass which are present as impurities. It is not possible to quantify the compounds by this method.
Identification and quantification of a multitude of or all individual components in a substance mixture without pure substances being available even today still constitutes an unsolved problem in mass spectrometry.